The present invention relates to the chemical and pharmaceutical industry, and more particularly, to a method for preparing a polysaccharide derivative of fibrinolysin, having a prolonged thrombolytic activity and fit for use in medicine.
Fibrinolysin finds extensive application in medicine as a thrombolytic preparation in the therapy of such diseases as myocardial infarction, thromboses and thromboembolism of lung arteries. The preparation is administered intravenously during several days. However, the fibrinolysin therapy is made complicated by the following factors. First, fibrinolysin does not have substrate specificity. It hydrolyzes not only fibrin, but other proteins as well, such as fibrinogen, casein and some proteins of the blood clotting system. Secondly, the application of fibrinolysin may cause not only an increase in the fibrinolytic activity, but also activation of the clotting system: as a result, secondary origination of clots may take place. Thirdly, as all enzymatic preparations, fibrinolysin becomes rapidly inactivated under physiological conditions due to the effect of pH, temperature, endogenic proteases and natural inhibitors; it is rapidly brought out of the blood flow so that it becomes necessary to introduce large doses of the enzyme during a long period of time. Moreover, treatment of patients with blood clots of considerable dimensions and with low anticoagulant characteristics of the blood proved to be unsuccessful.
Methods are known in the art for bonding enzymes by polymers which allow the attainment of preparations which, compared with native enzymes, have an essentially higher stability to various inactivating factors, longer residence in the organism and lower antigenicity. Thus, a method is known for preparing a derivative of fibrinolysin by covalently bonding fibrinolysin with a copolymer of ethylene and maleic anhydride at a temperature of from 0.degree. to 4.degree. C. over several hours. The resulting products have a biological activity of 15 to 30% (Zeitschrift fur Analytische Chemie, 1968, Band 243, No. 2, S.457).
The fibrinolysin derivative prepared by this method is incompatible with the tissues of the living organism, since the copolymer of ethylene with maleic anhydride is not biodegradable and cannot find application is medical practice.
Also known is a method of preparing a derivative of fibrinolysin with an aldehyde-containing polysaccharide which involves the reaction of fibrinolysin with a modified aldehyde-containing sephadex in a phosphate buffer at the temperature of 4.degree. C. during several hours. The non-bonded enzyme is washed-out by successive washings with a glacial phosphate buffer, 0.001 N solution of hydrochloric acid, 1 M solution of sodium chloride, with water and with a mixture of water and acetone, whereafter the preparation is dried with acetone (Kardiologiya, Moscow, 1977, No. 11, pp. 139-142).
The compound prepared by the above method is not soluble in water and aqueous solutions, this circumstance limiting its applicability for medical purposes.